Methanosarcina acetivorans utilizes a single NADPH-dependent thioredoxin system and contains additional thioredoxin homologues with distinct functions

Abstract

The thioredoxin system plays a central role in the intracellular redox maintenance in the majority of cells. The canonical system consists of an NADPH-dependent thioredoxin reductase (TrxR) and thioredoxin (Trx), a disulfide reductase. Although Trx is encoded in almost all sequenced genomes of methanogens, its incorporation into their unique physiology is not well understood. Methanosarcina acetivorans contains a single TrxR (MaTrxR) and seven Trx (MaTrx1-MaTrx7) homologues. We previously showed that MaTrxR and at least MaTrx7 compose a functional NADPH-dependent thioredoxin system. Here, we report the characterization of all seven recombinant MaTrxs. MaTrx1, MaTrx3, MaTrx4 and MaTrx5 lack appreciable disulfide reductase activity, unlike previously characterized MaTrx2, MaTrx6 and MaTrx7. Enzyme assays demonstrated that, of the MaTrxs, only the reduction of disulfide-containing MaTrx7 is linked to the oxidation of reduced coenzymes. NADPH is shown to be supplied to the MaTrxR-MaTrx7 system through the oxidation of the primary methanogen electron carriers F420H2 and ferredoxin, indicating that it serves as a primary intracellular reducing system in M. acetivorans. Bioinformatic analyses also indicate that the majority of methanogens likely utilize an NADPH-dependent thioredoxin system. The remaining MaTrxs may have specialized functions. MaTrx1 and MaTrx3 exhibited thiol oxidase activity. MaTrx3 and MaTrx6 are targeted to the membrane of M. acetivorans and likely function in the formation and the reduction of disulfides in membrane and/or extracellular proteins, respectively. This work provides insight into the incorporation of Trx into the metabolism of methanogens, and this reveals that methanogens contain Trx homologues with alternative properties and activities.

Fig. 1.

Insulin disulfide reductase activity of MaTrxs. Assays were performed in anaerobic buffer containing DTT (1 mM) alone or with the following: MaTrx1 (20 µM), MaTrx2 (10 µM), MaTrx3Δsp (20 µM), MaTrx4 (20 µM), MaTrx5 (20 µM), MaTrx6Δsp (5 µM) or MaTrx7 (5 µM). BDL, below detection limit.

Fig. 2.

Non-reducing SDS-PAGE of H₂O₂-oxidized MaTrxs. Each MaTrx_ox (10 µg) was separated by 15 % SDS-PAGE in the absence of a reducing agent.

Fig. 3.

CO-dependent reduction of NADP by M. acetivorans cell lysate. Assays were performed in sealed anaerobic cuvettes containing lysate (100 µg), NADP (0.5 mM) and a headspace of either N₂ or CO. Data points are the mean (±sd) of triplicate assays.

Fig. 4.

Analysis of disulfide isomerase and disulfide-forming activities of MaTrxs using scrambled and reduced RNase. (a) RNase activity of disulfide-scrambled RNase after incubation in GSH/GSSG redox buffer alone (control), with DsbA or with the indicated MaTrx. (b) RNase activity of reduced RNase after incubation in GSH/GSSG redox buffer alone (control), with DsbA or with the indicated MaTrx. Data points are the mean (±sd) of triplicate assays. **P≤0.002 (t-test).

Fig. 5.

Western blot analysis of fractionated cell lysates from M. acetivorans strains expressing FLAG-tagged MaTrx3 and MaTrx6. (a) Western blot analysis using anti-FLAG antibodies of membrane (M) and soluble (S) fractions of DJL80 cells containing MaTrx6-FLAG (18.8 kDa) and DJL81 cells containing MaTrx6Δsp-FLAG (15.8 kDa). Total protein in samples: M, 7.5 µg; S, 50 µg. (b) Western blot analysis using anti-FLAG antibodies of membrane (M) and soluble (S) fractions of DJL82 cells containing MaTrx3-FLAG (20.3 kDa) and DJL83 cells containing MaTrx3Δsp-FLAG (16.7 kDa). Total protein in samples: M, 5 µg; S, 35 µg.

Fig. 6.

Model showing the location and proposed function(s) of MaTrxs in M. acetivorans. Solid lines indicate detected activities and dashed lines indicate putative activities. Question marks denote unknown or proposed protein(s) or factor(s). MaTrx7 was previously shown to reduce disulfides in oxidized MsvR, activating binding of DNA [43]. Fd_o, oxidized ferredoxin; Fd_r, reduced ferredoxin; Fnr, ferredoxin : NADP oxidoreductase; Fno, F₄₂₀H₂ : NADP oxidoreductase.